Urodeles including newt and salamander have remarkable regenerative capacity during the postembryonic life span. Some of the unique features are the formation of the well-developed wound epidermis and the active dedifferentiation process in the early phase of regeneration. These are regarded as key events for the successful regeneration since no further regenerative activity is possible without them. In this study, we investigated the role of retinoic acid (RA) in salamander limb regeneration by blocking RA synthesis using disulfiram, an inhibitor of aldehyde dehydrogenase that oxidizes retinal to RA. Disulfiram treatment resulted in delaying the limb regeneration processes via inhibition of wound epidermis formation and dedifferentiation process. When RA was administered after disulfiram treatment, the inhibitory effect of disulfiram was rescued. In addition, disulfiram treatment after the dedifferentiation stage resulted in the mild retardation of limb regeneration, suggesting that RA might also be involved in the blastema outgrowth. Furthermore, salamander MMP-9 gene expression was also inhibited by disulfiram treatment. Collectively, our findings indicate that endogenous RA may play an important role(s) in the early phase of limb regeneration by regulating the expression of molecules responsible for the modification of intracellular and extracellular environment during salamander limb regeneration.